Enzyme assay with duplicate fluorophores

ABSTRACT

Compositions and methods are disclosed that provide a rapid, sensitive, and accurate cell-based assay for enzyme activity, particularly for enzyme activities associated with  botulinum  toxins. A cell is provided that expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

This application claims the benefit of U.S. Provisional Application No.61/895,533 (filed Oct. 25, 2013), U.S. Provisional Application No.61/897,352 (filed Oct. 30, 2013), U.S. Provisional Application No.62/014,586 (filed Jun. 19, 2014), and U.S. Provisional Application No.62/058,532 (filed Oct. 1, 2014).

FIELD OF THE INVENTION

The field of the invention is protease assays, especially those relatedto Clostridium botulinum neurotoxins.

BACKGROUND

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, andare among the most potent toxins known. These toxins are awell-recognized source of food poisoning, often resulting in seriousharm or even death of the victims. There are a number of structurallysimilar botulinum neurotoxins or serotypes (BoNT/A-G, and a proposedBoNT/H), each of which is composed of a heavy chain (˜100 kD) and alight chain (˜50 kD). The heavy chain mediates toxin entry into a targetcell through receptor-mediated endocytosis. Once internalized, the lightchain is translocated from the endosomal vesicle lumen into the cytosol,and acts as a zinc-dependent protease to cleave substrate specificproteins that mediate vesicle-target membrane fusion, a process that iscentral to neurotransmitter release.

BoNT substrate proteins include the cell membrane protein syntaxin,peripheral membrane protein SNAP-25, and the vesicular membrane proteinsynaptobrevin (Syb). These proteins are collectively referred to asSNARE (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor) proteins. Cleavage of SNARE proteins blocks vesicle fusionwith the cell membrane and abolishes neurotransmitter release atneuromuscular junctions. Among the SNARE proteins, syntaxin and SNAP-25usually reside on the target membrane and are thus referred to ast-SNAREs, while synaptobrevin is associated exclusively with synapticvesicles within the synapse and is referred to as a v-SNARE. Together,these three proteins form a complex that is thought to be the minimalmachinery needed to mediate fusion between vesicle membrane and plasmamembrane. BoNT/A, E, and C cleave SNAP-25, whereas BoNT/B, D, F, and Gcleave synaptobrevin (Syb) at separate and distinct sites. BoNT/C alsocleaves syntaxin in addition to SNAP-25. Since BoNTs act as enzymes,even minute quantities can have a devastating effect on an affectedindividual.

While botulinum toxin is a source of food poisoning and has thepotential for use as a bioterrorism weapon, there are therapeuticapplications. Recently, botulinum toxin has been utilized to treatconditions associated with unwanted muscle contractions (such asstrabismus) and in the treatment of persistent migraines. It is alsowidely used for cosmetic purposes, where the selective paralysis ofsmall muscles beneath the skin temporarily reduces the appearance ofage-related wrinkles. With such widespread use there is a need tosensitively and speedily characterize BoNT proteins. This process iscomplicated by the need to accurately quantify BoNT activity rather thansimply quantify the amount of BoNT protein present, as purificationprocesses utilized in isolating these proteins can lead to a significantdegree of denaturation and resulting inactivation of these proteins.

Currently, a commonly used method to detect BoNTs and quantify theiractivity is to perform toxicity assays using mice. Such methods requirethe use of large numbers of mice, are time-consuming, and cannot be usedto study toxin catalytic kinetics. A number of immunoassay systems basedon antibodies developed against BoNT proteins have also been developed,but while such assays may be useful for quantifying the amount of BoNTprotein present they cannot be used to determine the toxin's enzymaticactivity. Methods have been developed to detect BoNT reaction productsin order to measure enzymatic activities of these toxins, for example,using HPLC or immunoassays directed to cleavage products. These methods,however, are generally complex, time-consuming, and can be expensive(for example, utilizing specialized antibodies), making them difficultto automate and inapplicable for large-scale screening.

Recently, researchers have begun exploring the use of fluorescenceresonance energy transfer (FRET) methods for quantifying enzymaticactivities. FRET methods involve the use of two fluorescent moieties, adonor fluorophore and an acceptor fluorophore. The emission spectrum ofthe donor fluor overlaps the excitation spectrum of the acceptor fluor,and under defined conditions and at proper fluorophore spacing andorientation excitation of the donor fluor can lead to emission from theacceptor fluor. The efficiency of this energy transfer is highlydependent upon the distance between the donor fluor and the acceptorfluor, and numerous fluorescence assays have been developed to exploitthis phenomenon. For application in cell-based assays, such FRET probescan be generated within the cell by genetic manipulation. In such anapproach fluorescent proteins, in particular Green Fluorescent Proteinand variants thereof as described in International Patent ApplicationWO2008/145301A1 (to Tasdemir and Corazza), are often used as theseproteins do not require the addition of a cofactor or substrate in orderto fluoresce. Some of these assays are capable of detecting enzymaticactivity. For example, U.S. Pat. No. 7,749,759 (to Fernandez-Salas,Steward, and Aoki) discloses the use of cells containing a substrate fora Clostridium toxin, where the substrate (which is expressed from agenetic construct) includes a donor fluorophore and an acceptorfluorophore separated by a peptide that is cleaved by the Clostridiumtoxin. Exposure to the Clostridium toxin results in cleavage of thesubstrate, and the subsequent separation between the donor fluorophoreand the acceptor fluorophore results in changes in the observedfluorescence. Such FRET-based assays, however, have limitations. Theexcitation spectra of the donor fluorophore and of the acceptorfluorophore frequently overlap, resulting in an inherently highbackground signal from the acceptor fluorophore even in the absence ofFRET. Similarly, in some reporter constructs the fluorophores may selfaggregate, forming fluorophore complexes within and/or betweenaggregated constructs that do not dissociate on cleavage of a targetsite. In addition, the use of longer peptide sequences as cleavage sitesin order to accommodate more complex enzyme binding and cleavage sites(for example, those of Clostridial neurotoxins) can dramatically reducethe efficiency of energy transfer between fluorophores separated by suchpeptide sequences.

As a result of this low efficiency and high background fluorescence,FRET-based constructs are often overexpressed within cells, resulting inundesirable cell toxicity and construct aggregation. U.S. Pat. No.6,936,428 (to Davis and Vellencourt) describes an approach in whichbackground fluorescence in FRET constructs that utilize donor andacceptor fluorescent proteins is reduced by using constructs in whichpairs of multimeric protein fluorophores are positioned to formintramolecular homodimers, thereby reducing the formation ofdonor/acceptor heterodimers that generate background fluorescence.Alternatively, U.S. Pat. No. 8,067,231 (to Fernandez-Salas et al)describes a cell-based assay in which a change in the distribution ofobservable fluorescence from a cell membrane to the cell cytoplasmicspace is observed, however such characterization requires sophisticatedoptical instruments and image analysis.

All other publications referenced herein are incorporated by referenceto the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Thus, improved compositions and methods are therefore needed to providerapid and accurate characterization of BoNTs and BoNT activities.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for use inassays that detect botulinum neurotoxins (BoNTs) which utilize aconstruct that includes at least two instances of the same reporter,such as a fluorophore or chromophore. The construct includes ananchoring site that attaches the construct to a cell and/or vesiclemembrane, which in turn provides protection of the reporter from adegradative activity in the cytosol. The construct also includes acleavage site that separates the anchoring site from at least one of thereporters and also serves as a substrate for an enzyme activity (forexample, protease activity). Enzyme activity at the cleavage sitereleases reporter from the construct; subsequent degradation of thereporters in the cytosol results in a measureable change from a baselinesignal that is proportional to enzyme activity.

Embodiments of the inventive concept include a reporting construct thatcan be used for characterizing an enzyme activity (for example, theactivity of a botulinum neurotoxin), and cells that include a nucleicacid encoding for such a reporting construct. The reporting constructincludes a membrane anchoring domain that interacts with a membrane of acell (for example, a plasma membrane or a vesicle membrane), a reporterdomain that includes two or more occurrences of a signal generatingpeptide (for example, a peptide sequence corresponding to a fluorescentprotein or having at least 80% sequence identity to Green FluorescentProtein), and a cleavage site that is located between the membraneanchoring site and the reporter domain. The signal generating peptidesproduce indistinguishable signals, and the total signal produced by thereporter domain is an aggregate of these individual yetindistinguishable signals. The cleavage site includes a peptide that issusceptible to cleavage by the enzyme activity (for example a SNAREprotein or a fragment thereof), and such cleavage results in the releaseof the reporter domain into the cytoplasm. Such cleavage sites can beselected to be susceptible to cleavage by more than one enzyme (forexample, both BoNT/A and BoNT/E). The reporter domain undergoesdegradative events following release into the cytoplasm, with separatedegradative events causing a loss of signal from each of the signalgenerating peptides and resulting in a gradual loss of the aggregatesignal of the reporter domain. In some embodiments a linker peptide isinterposed between the signal generating peptides. In a preferredembodiment of the inventive concept, the construct can be cleaved bymore than one enzyme, and shows reduced bias in regards tosusceptibility of cleavage by one enzyme activity (for example BoNT/A)over a second enzyme activity (for example BoNT/E) relative to ananalogous construct having a reporting domain with a single signalgenerating peptide. In some embodiments the reporting construct includesan auxiliary reporting domain that provides a signal that isdistinguishable from that of the reporter domain.

Another embodiment of the inventive concept is a method forcharacterizing an enzyme activity by providing a cell that expresses areporting construct, contacting the cell with a sample suspected ofincluding the enzyme activity, and observing a decrease in a signalgenerated by the reporting construct in the presence of the enzymeactivity. The reporting construct includes a membrane anchoring domainthat interacts with a membrane of a cell (for example, a plasma membraneor a vesicle membrane), a reporter domain that includes two or moreoccurrences of a signal generating peptide (for example, a peptidesequence corresponding to a fluorescent protein and/or having at least80% sequence identity to Green Fluorescent Protein), and a cleavage sitethat is located between the membrane anchoring site and the reporterdomain. The signal generating peptides produce indistinguishablesignals, such that the total signal produced by the reporter domain isan aggregate of these individual yet indistinguishable signals. Thecleavage site includes a peptide that is susceptible to cleavage by theenzyme activity (for example a SNARE protein or a fragment thereof), andsuch cleavage results in the release of the reporter domain into thecytoplasm. The reporter domain undergoes degradative events followingrelease into the cytoplasm, with separate degradative events causing aloss of signal from each of the signal generating peptides and resultingin a gradual loss of the aggregate signal of the reporter domain. Insome embodiments the aggregate signal of the reporter domain isobservable following the loss of the signal from one of the signalgenerating peptides from a degradative event. In a preferred embodimentof the inventive concept, the construct can be cleaved by more than oneenzyme, and shows reduced bias in regards to susceptibility of cleavageby one enzyme activity (for example BoNT/A) over a second enzymeactivity (for example BoNT/E) relative to an analogous construct havinga reporting domain with a single signal generating peptide.

In some methods of the inventive concept a reference signal is providedthat is distinguishable from the signal provided by the reporter domainand can be utilized for normalization of the signal from the reporterdomain of the reporting construct. In some embodiments the referencesignal is provided by including an auxiliary reporter with theconstruct, where the auxiliary reporter produces a signaldistinguishable from the reporter domain and is not affected by theenzyme activity In other embodiments the reference signal is provided bycontacting the cell with a cell dye (for example a membrane dye or anucleus/nuclear dye). Such a cell dye can be brought into contact withthe cell before, during, or after contacting the cell with the enzymeactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D schematically depict constructs of the inventive conceptand show typical results. FIGS. 1A to 1C schematically depict constructsof the inventive concept that have two identical reporting peptides invarious configurations. FIG. 1D shows typical results for a cell-basedassay for two different botulinum neurotoxins (BoNT/A, BoNT/E),utilizing a reporter configured as shown in FIG. 1A. Results are alsoshown for cells expressing an analogous construct carrying a singlereporter. Surprisingly, the bias between BoNT/A and BoNT/E sensitivityis dramatically reduced in cells expressing the construct of theinventive concept.

FIGS. 2A to 2K schematically depict constructs of the inventive conceptthat include two identical reporters and a third, different reporter.

FIG. 3 depicts a cell-based assay of the invention in which theconstruct includes a cell membrane anchoring domain.

FIG. 4 depicts a cell-based assay of the invention in which theconstruct includes a vesicle membrane anchoring domain.

FIG. 5 shows an assay methodology of the invention.

FIG. 6 shows an alternative assay methodology of the invention.

FIG. 7 shows excitation and emission spectra for a fluorescentprotein(eYFP) and a secondary dye,

FIG. 8 schematically depicts an assay method of the inventive conceptthat incorporates the use of a secondary dye.

FIGS. 9A and 9B shows exemplary data from an assay of the inventiveconcept, in the absence of correction and with data normalization usinga secondary dye, respectively.

FIG. 9C shows photomicrographs produced using fluorescence and brightfield microscopy of cells expressing a construct of the inventiveconcept, in the absence and in the presence of a corresponding botulinumtoxin.

DETAILED DESCRIPTION

Embodiments of the inventive concept utilize one or more cells thatinclude a construct (for example introduced via expression followingtransfection and/or microinjection) that includes components thatsequester an observable reporter moiety or region in a protected regionand an analyte-sensitive region. The reporter moiety can include two ormore identical reporters. Such protected regions can be in proximity toa cell membrane and/or a vesicle membrane. Interaction of the analytesensitive region with the analyte results in the release of the reporterfrom the protected region by a cleavage event, which results in thedegradation of the reporter and an observable change in the signal fromthe reporter. Examples of analytes can include proteolytic enzymes, andin such cases the analyte sensitive region can be a cleavage site thatcan serve as an enzyme substrate.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

Embodiments of the inventive concept include methods in which suchconstructs are expressed in cells, which are subsequently exposed to ananalyte of interest (for example, a Clostridium botulinum and/orClostridium tetani toxin). Such cells can be division arrested cells. Itshould be appreciated that the novel arrangement and composition ofconstructs of the inventive concept has a direct impact on the mechanismand performance characteristics of such assays. In preferred embodimentsof the inventive concept exposure to the analyte of interest results ina cleavage event at an analyte sensitive region or domain of theconstruct, resulting in the release of a construct fragment thatincludes a reporter region carrying two or more identical signalgenerating regions (for example a pair of identical fluorophores) eachof which generate a detectable signal. Unlike constructs and methodsutilizing fluorophores arranged as FRET pairs (for example, differentfluorophores arranged as hetero-FRET pairs and/or similar or identicalfluorophores arranged as homo-FRET pairs), each of the signal generatingregions of a reporter released as a construct fragment contributesdirectly to a detectable aggregate signal both before and immediatelyfollowing the cleavage event. For example, in some embodiments such anaggregate signal can be an approximate summation (or other function) ofthe signal observed from each of the signal generating regions. Inconstructs and assays of the inventive concept the change in thedetectable signal that forms the basis of detection is a result ofmultiple degradation events that occur in the cytosol subsequent to thecleavage event, as loss of one of a pair of identical signal generatingregions (for example, a pair of essentially identical fluorophores)still providing an emitting reporter fragment. In a construct utilizinga single reporter, a single degradation event occurring at a releasedconstruct fragment can result in fragmentation of the single reporterand loss of the detectable signal. Similarly, in constructs utilizingduplicate reporters in which the detectable signal is a result ofinteraction between the reporters (for example, a construct utilizing apair of fluorophores arranged to perform hetero-FRET and/or homo-FRET)the fragmentation of a single signal generating region due to a singledegradation event also results in a loss of the detectable signal fromthat construct.

In contrast, a reporter-containing fragment generated by a cleavageevent directed to a construct of the inventive concept that isconfigured to release two or more identical/indistinguishable signalgenerating regions following the cleavage event can continue to providea detectable signal following a single degradation event occurring atone of the occurrences of the signal generating region. Since multipledegradation events are required to halt the generation of detectablesignal by the construct cleavage fragment, the detectable signaldecreases with increasing analyte concentration but persists (relativeto single reporter constructs or constructs that depend upon pairedreporters for signal generation) at high analyte concentrations. Thiscan result in an improved dynamic range for assays based on constructsof the inventive concept.

The components of a construct of the inventive concept can be arrangedin a variety of ways. The following description includes a number ofexamples in which functional domains of a reporting construct, forexample, a membrane anchor (A), a cleavage site (B), multipleoccurrences of a primary signal generating region/reporter (C, C′), andin some instances a secondary reporter (D) are depicted as being joinedin various arrangements by linker regions or linkers (−). It should beappreciated that, in the following figures and their descriptions, thepresence of these linkers can be considered optional. As such, inembodiments of the inventive concept one or more portions described as alinker can be omitted. In some embodiments linker regions of theinventive concept can also include portions of a functional region thatare not directly involved in the function of that region. For example,if a reporter is a protein fluorophore a linker can be a portion of theprotein fluorophore sequence that is not directly involved influorescence. Similarly, a linker can be a portion of cleavage sitesequence that does not directly serve as a protease substrate.Alternatively, in other embodiments of the inventive concept a linkercan be a synthetic or engineered peptide sequence, which can, forexample, be designed to reduce FRET (i.e. homo-FRET and/or heter-FRET)between signal generating regions to non-useful levels (for example,less than about 5%). Such a synthetic peptide sequence can be a flexiblesequence, a rigid sequence, or a sequence with both flexible and rigidportions. SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ IDNO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 showexemplary synthetic linker sequences. In some embodiments a linkerregion can include repeated occurrences of such linker sequences (forexample, in a concatemer-like arrangement) to provide desired length,flexibility, and/or other desirable structural features. It should beappreciated that, as used herein, the term “linker” does not denote acleavage site that is cleaved by an enzyme analyte, but rather astructural region that joins other functional regions of a reportingconstruct.

In some embodiments the reporter-containing region can contain at leasttwo instances of a primary reporter that is a fluorophore and/or achromophore. In some embodiments of the inventive concept, two instancesof a primary reporter have the same amino acid sequence. In otherembodiments, two instances of a primary reporter can have differentcompositions but have substantially similar (i.e. exhibiting greaterthan or equal to 80% overlap) excitation and emission spectra. Inpreferred embodiments a primary reporter can be a fluorescent protein,for example Green Fluorescent Protein (SEQ ID NO. 10) or a peptidehaving at least 80% sequence identity to the sequence of GreenFluorescent Protein. Suitable fluorescent protein fluorophores includeYellow Fluorescent Protein (for example eYFP, SEQ ID NO. 11), RedFluorescent Protein, Cyan Fluorescent Protein (SEQ ID NO. 12), mBanana,mStrawberry, mCherry, tdTomato, J-Red, DsRed monomer, mCitrine, Venus(SEQ ID NO. 13), YPet protein, Emerald, EGFP, CyPet, mCFPm, Cerulean,mPlum, mOrange, mKO, T-Sapphire, a derivative of Yellow FluorescentProtein, a derivative of mCitrine, a derivative of Venus, a derivativeof YPet protein, and/or a Green Fluorescent Protein variant. In anespecially preferred embodiment, a primary reporter can be a monomericfluorescent protein derived from the Green Fluorescent Protein ofAequorea victoria, such as Sirius, Azurite, EBFP2, TagBFP, mTurqoise,ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EDFP,mWasabi, EmGFP, TagYFP, eYFP (SEQ ID NO. 11), Topaz, SYFP2, Venus,Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry,mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire,mAmetrine, and/or mKeima (see“Fluorescent Proteins and TheirApplications in Imaging Living Cells and Tissues” (Chudakov, D. M. etal, Physiol. Rev. 90:1103-1163, 2010). Similarly, suitable primaryreporters can be protein fluorophores derived from the Green FluorescentProtein of Aequorea victoria that include an A206K mutation.Alternatively, a primary reporter can be a non-fluorophore and/or anon-chromophore, for example a fluorescence quencher.

Within constructs of the inventive concept the arrangement of primaryreporters within the reporter containing portion can be such that theyare sufficiently distant from one another and/or oriented such that theyexhibit essentially no useful (i.e. less than 5%) FRET (for example,homo-FRET) energy transfer. Contemplated low levels of homo-FRET energytransfer can be less than or equal to about 1%, less than or equal toabout 0.1%, less than or equal to about 0.01%, and/or less than or equalto about 0.001% energy transfer between fluors. Similarly, contemplatedlow levels of homo-FRET energy transfer can be less than or equal toabout 10%, less than or equal to about 1%, less than or equal to about0.1%, less than or equal to about 0.01%, and/or less than or equal toabout 0.001% of the background noise of the observable signal.Alternatively, in some embodiments of the inventive construct theprimary reporters 120, 130 can be arranged such that they exhibitsignificant (i.e. greater than about 1%) homo-FRET energy transfer. Suchphenomena can be controlled using the length of a linker region orlinker interposed between such primary reporters. In some embodiments ofthe inventive concept such a linker can have a length of 20, 30, 40 50,or more amino acids. Similarly, the such a linker can have a lineardimension of at least about 4, about 6, about 8, about 10, about 15,about 20 or more nanometers when the construct is in its native, foldedstate.

It is contemplated that a primary reporter of a construct of theinventive concept can include more than one fluorescent moiety. Forexample, a pair of fluorophores with different but overlappingexcitation and emission spectra could be arranged as a FRET pair thatacts as a single instance of a primary reporter. Similarly, a pair ofidentical fluorophores could be arranged as a homo-FRET pair that actsas a single instance of a primary reporter (for instance, as detected byfluorescence anisotropy). For example, in such an embodiment a reportingconstruct could include a pair of primary fluorophores, where eachprimary fluorophore includes two fluorophores with different butoverlapping excitation and emission spectra arranged as a hetero-FRETpair. Alternatively, in such an embodiment a reporting construct couldinclude a pair of primary fluorophores, where each primary fluorophoreincludes two fluorophores with similar or identical excitation andemission spectra arranged as a homo-FRET pair.

As noted above, the domains of a construct of the inventive concept canbe arranged in a variety of ways. A preferred embodiment of theinventive concept, which can be characterized as an A-B-C-C′arrangement, is shown schematically in FIG. 1A. Such a reporterconstruct can include a membrane anchor 170 that is linked to a cleavagesite 160 by an interposing anchor/cleavage site linker 150. The cleavagesite is in turn linked to a first instance of a pair of identicalprimary reporters 120 by a cleavage site/reporter linker 165. This firstinstance of a pair of identical primary reporters 120 is linked to asecond instance of a pair of identical primary reporters 130 by aninterposing primary reporter/primary reporter linker 140. In such anembodiment the reporter-containing region can contain at least twoinstances of a primary reporter. An example of a reporter constructhaving such a structure is shown in SEQ ID NO 14.

An alternative arrangement of the reporter construct (which can becharacterized as A-C-B-C′) is shown schematically in FIG. 1B, in which amembrane anchor 170 is linked to a first instance of a pair of identicalprimary reporters 120 by an interposing anchor/primary reporter linker125. The first instance of a pair of identical primary reporters 120 islinked to a cleavage site 160 via a first cleavage site/fluor linker165A. The cleavage site 160 is also linked to a second instance of apair of identical primary reporters 130 via a second cleavage site/fluorlinker 165B. Emission from such a retained reporter 120 can, forexample, be used as a baseline or normalizing signal.

Another alternative arrangement of the reporter construct (which can becharacterized as C-A-B-C′) is shown in FIG. 1C, in which a firstinstance of a pair of identical primary reporters 120 is linked to amembrane anchor portion 170 by an intervening anchor/primary reporterlinker 125. The membrane anchor 170 is also linked to a cleavage site160 by an anchor/cleavage site linker 150. The cleavage site is 160 is,in turn, linked to a second instance of a pair of identical primaryreporters 130 via a cleavage site/reporter linker 165. Emission fromsuch a retained reporter 120 can, for example, be used as a baseline ornormalizing signal. Although one representation of this configuration isshown, it should be appreciated that the reporter linker 140 can beplaced on either side of the cleavage site 160 in such an embodiment.

In the configurations for the construct shown in FIG. 1A, FIG. 1B, andFIG. 1C hydrolysis of the cleavage site 160, for example by a protease,results in the release of one or more primary reporters 120, 130 fromthe membrane anchoring domain 170. The cleavage site, therefore, atleast partially determines the specificity of assays based upon suchconstructs for specific enzyme activities, and preferably includeshydrolysis sites where enzyme activity results in cleavage of thepeptide backbone of the construct and recognition sites that provideinteraction sites with the enzyme and confer at least a portion ofsubstrate specificity. In some embodiments the cleavage site can includeregions that interact with exosites or allosteric sites of a targetenzyme. In preferred embodiments of the inventive concept the cleavagesite is susceptible to cleavage by a Botulinum neurotoxin BoNT proteaseactivity, for example BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F,and/or BoNT/G. It is contemplated that a cleavage site sequence can beselected or designed to provide cleavage and recognition sites for twoor more different BoNTs (for example, both BoNT/A and BoNT/E). In someembodiments the cleavage site can include intact sequences or sequencesderived from a SNARE protein, such as a synaptosomal-associated protein(for example, SNAP-25; SEQ ID NO. 10), a vesicle associated membraneprotein (for example, VAMP or synaptobrevin; SEQ ID NO. 11), and/orsequences derived from a syntaxin. Examples of suitable cleavage sitesequences include SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and SEQID NO. 18.

As noted above, release from a membrane anchoring domain can permit oneor more primary reporters to diffuse away from a protected region. Suchprotected regions can include proximity to a cell membrane and/or to avesicle membrane. Towards that end an anchoring domain can include acell membrane localizing peptide and/or a vesicle membrane localizingpeptide. Such an anchoring region can include a transmembrane region inwhich at least a portion of the peptide chain enters and/or passesthrough a membrane of the cell. Alternatively, an anchoring region caninclude a palmitoylation site that, following post-translationalprocessing by the cell, provides a site that interacts with a membraneof the cell. Alternatively, a membrane anchoring site can include apeptide sequence that interacts with a membrane-bound receptor orligand. In some embodiments of the inventive concept an anchoring domaincan include sequences derived from a synaptobrevin (for example atransmembrane peptide of synaptobrevin) and/or from SNAP-25 (for examplea palmitoylation region of SNAP-25). In other embodiments of theinventive concept a membrane anchoring domain can be a syntaxin or asyntaxin fragment.

As noted above, once released from an anchoring region 170, primaryreporters can diffuse into the cytosol. Use of two or more primaryreporters provides a number of technical advantages. Advantages ofconstructs that release two or more primary reporters upon cleavage aredescribed above. Other advantages include the ability to utilize lowerexpression levels for the construct (thereby minimizing the risk oftoxicity and/or construct aggregation) and/or the ability to utilize farfewer cells in a cell-based assay. In addition, the relatively strongsignal generated by multiple primary reporters can provide for morestable and reproducible baseline measurements, which also serve toincrease assay sensitivity by permitting more robust differentiation ofsignal from noise. It should be noted that previous investigators (A. G.von Armin, X. W. Deng, and M. G. Stacey; Gene 221 (1998), pp. 35-43)have utilized constructs incorporating two or more sets of YellowFluorescent Protein sequences as markers for gene expression but foundbright fluorescence throughout the cells, such constructs failing todemonstrate the degradative events that are not only characteristic ofconstructs of the inventive concept but that are necessary for theirutilization in cell-based assays.

Surprisingly, the inventors have found that constructs with reportingregions that include multiple primary reporters can provide differentselectivity between enzyme targets compared to analogous constructs inwhich the reporting region contains a single occurrence of a primaryreporter. An example of this is found in FIG. 1D, which showscomparative data for the response of cell based assays to BoNT/A and toBoNT/E, where the cells include either a construct of the inventiveconcept that includes two eYFP protein fluorophores in an A-B-C-C′arrangement or an analogous construct that includes a single eYFPprotein fluorophore in an A-B-C arrangement. Both BoNT/A and BoNT/Erecognize and cleave the cleavage site of both constructs, however thesingle fluorophore construct has an approximately 80-fold bias (i.e.approximately 80-fold better sensitivity, as expressed in terms of EC₅₀)for the BoNT/A neurotoxin over the BoNT/E neurotoxin (see FIG. 1D, 37°C.). The same study performed using cells expressing the “dualfluorophore” reporting construct (i.e. the construct carrying two eYFPfluorophores) showed similar sensitivity to BoNT/A but increasedsensitivity to BoNT/E, reducing the assay bias to 26-fold. Thisreduction in assay bias advantageously permits the same construct to beused for assays directed towards more than one enzyme analyte. Withoutwishing to be bound by theory, the inventors believe that the use ofadditional fluorescent peptide sequences imparts a tertiary structure tothe construct that provides improved access to a greater number ofrecognition sites within the cleavage domain of the construct.

In some embodiments of the inventive concept a reporter construct caninclude, in addition to two or more primary reporters, a secondaryreporter that differs from the primary reporters. The secondary reportercan have a distinct or different chemical structure from that of theprimary reporters. Such a secondary reporter can have an emissionspectrum that overlaps the excitation spectrum of a primary reporter. Insuch an embodiment the construct can be arranged so that significantFRET (i.e. greater than about 1%) occurs between a secondary reporterand a primary reporter. In a preferred embodiment the secondary reporteris a fluorescent protein, such as Yellow Fluorescent Protein, RedFluorescent Protein, Cyano Fluorescent Protein, Green FluorescentProtein, mBanana, mStrawberry, mCherry, tdTomato, J-Red, DsRed monomer,mCitrine, Venus, YPet protein, Emerald, EGFP, CyPet, mCFPm, Cerulean,mPlum, mOrange, mKO, T-Sapphire, a derivative of Yellow FluorescentProtein, a derivative of mCitrine, a derivative of Venus, a derivativeof YPet protein, and/or a Green Fluorescent Protein variant.

Preferably, the reporter construct can be arranges such that significantor useful FRET does not occur (i.e. the degree of FRET that occurs isless than or equal to 5%) between the secondary reporter and a primaryreporter. The arrangement of the secondary reporter and at least one ofthe primary reporters within the reporter construct can be such thatthey are sufficiently distant from one another that they exhibitessentially no useful (i.e. less than or equal to 5%) FRET. Contemplatednon-useful levels of FRET energy transfer can be less than or equal toabout 10%, less than or equal to about 5%, less than or equal to about1%, or less than or equal to about 0.1% of the associated backgroundfluorescence. This can be accomplished by selecting a linker thatprovides sufficient distance between the secondary reporter and aprimary reporter. In some embodiments of the inventive concept such alinker can have a length of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 ormore amino acids. Similarly, the a linker between two primary reporterscan have a length of at least about 4, about 6, about 8, about 10, about15, about 20 or more nanometers when the construct is in its native,folded state. Suitable linkers can include synthetic peptides, and suchpeptides can be flexible peptides, rigid peptides, or can include bothflexible and rigid portions.

In some embodiments of the inventive concept a signal or emission from asecondary reporter can be utilized as a reference or as normalizationdata useful for adjusting or normalizing a signal observed from one ormore reporters of the reporter-containing portion, thereby improvingprecision and/or sensitivity of an assay utilizing such a construct. Inother embodiments a signal or emission from a secondary reporter can beutilized by an image recognition system to identify the location withinan acquired image wherein a reaction of the assay can be taking place,thereby simplifying data acquisition.

An example of an embodiment that includes such a secondary reporter(which can be characterized as having the structure A-D-B-C-C′, where“D” represents the secondary reporter) is shown schematically in FIG.2A. In such a reporter construct a membrane anchor 270 is linked to asecondary reporter 280 by an intervening anchor/secondary reporterlinker 285. The secondary reporter 280 is also linked to a cleavage site260 by a secondary reporter/cleavage site linker 255. The cleavage site260 is, in turn, linked to a first instance of a pair of identicalprimary reporters 220 via a cleavage site/primary reporter linker 265,and the first instance of a pair of identical primary reporters 220 andthe second instance of a pair of identical primary reporters 230 arejoined by an intervening primary reporter/primary reporter linker 240.

An alternative embodiment of a reporting construct with two or moreprimary reporters and at least one secondary reporter (which can bedescribed as D-A-B-C-C′) is depicted schematically in FIG. 2B. In thisembodiment a secondary reporter 280 is coupled to a membrane anchor 270by an intervening anchor/secondary reporter linker 285. The membraneanchor 270 is in turn linked to a cleavage site 260 by ananchor/cleavage site linker 250. The pair of identical primary reporters220, 230, which are joined by a primary reporter/primary reporter linker240 are in turn attached to the cleavage site 260 via a cleavagesite/primary reporter linker 265.

FIG. 2C schematically depicts an embodiment of a reporting construct(which can be described as C-D-A-B-C′) in which a first instance of apair of identical primary reporters 220 is joined to a secondaryreporter 280 by a primary reporter/secondary reporter linker 275. Thesecondary reporter 280 is in turn coupled to a membrane anchor 270 by anintervening anchor/secondary reporter linker 275. The membrane anchor270 is further joined to a cleavage site 260, with an anchor/cleavagesite linker 250 interposed between them. A second instance of a pair ofidentical primary reporters 230 is also coupled to the cleavage site 260by a cleavage site/primary reporter linker 265.

FIG. 2D schematically depicts an embodiment of a reporting construct(which can be described as D-C-A-B-C′) in which a secondary reporter 280is joined to a first instance of a pair of identical primary reporters220 by a primary reporter/secondary reporter linker 275. The firstinstance of a pair of identical primary reporters 220 is in turn coupledto membrane anchor 270, with an anchor/primary reporter linker 245interposed between them. The membrane anchor 270 is also joined to acleavage site 260 by an anchor/cleavage site linker 250. A secondinstance of a pair of identical primary reporters 230 is coupled to thecleavage site 260 by an interposing cleavage site/primary reporterlinker 240.

FIG. 2E schematically depicts an embodiment of a reporting construct(which can be described as A-D-C-B-C′) in which a membrane anchor 270 isjoined to a secondary reporter 280 via an anchor/secondary reporterlinker 285. The secondary reporter 280 is also coupled to a firstinstance of a pair of identical primary reporters 220 by an interveningprimary reporter/secondary reporter linker 275. The first instance of apair of identical primary reporters 220 is in turn coupled to a cleavagesite 260 by a cleavage site/primary reporter linker 265A. The secondinstance of a pair of identical primary reporters 230 is also joined tothis cleavage site 260 by another cleavage site/primary reporter linker265B.

FIG. 2F schematically depicts an embodiment of a reporting construct(which can be described as A-C-D-B-C′) in which a membrane anchor 270 isjoined to a first instance of a pair of identical primary reporters 220via an interposing anchor/primary reporter linker 245. A secondaryreporter 280 is also coupled to the first instance of a pair ofidentical primary reporters 220 via a primary reporter/secondaryreporter linker 275. This secondary reporter 280 is linked to a cleavagesite 260 by a secondary reporter/leavage site linker 255. Subsequently,the cleavage site 260 is joined to a second instance of a pair ofidentical primary reporters 230 by a cleavage site/primary reporterlinker 265.

FIG. 2G schematically depicts an embodiment of a reporting construct(which can be described as A-C-B-C′-D) in which a membrane anchor 270 iscoupled to a first instance of a pair of identical primary reporters 220by an anchor/primary reporter linker 245. This first instance of a pairof identical primary reporters 220 is joined to a cleavage site 260 viaa first cleavage site/primary reporter linker 265A. The cleavage site260 is also joined to a second instance of a pair of identical primaryreporters 230 by a second cleavage site/primary reporter linker 265B. Asecondary reporter 280 is also coupled to the second instance of a pairof identical primary reporters 230 by an intervening primaryreporter/secondary reporter linker 275.

FIG. 2H schematically depicts an embodiment of a reporting construct(which can be described as A-C-B-D-C′) in which a membrane anchor 270 iscoupled to a first instance of a pair of identical primary reporters 220by an anchor/primary reporter linker 245. This first instance of a pairof identical primary reporters 220 is joined to a cleavage site 260 viaa cleavage site/primary reporter linker 265. The cleavage site 260 isalso joined to a secondary reporter 280 by a secondary reporter/cleavagesite linker 255. A second instance of a pair of identical primaryreporters 230 is also coupled to the secondary reporter 280, by anintervening primary reporter/secondary reporter linker 275.

FIG. 2I schematically depicts an embodiment of a reporting construct(which can be described as A-B-C-D-C′) in which a membrane anchor 270 iscoupled to a cleavage site 260 by an anchor/cleavage site linker 250.This cleavage site 260 is joined to a first instance of a pair ofidentical primary reporters 220 via a cleavage site/primary reporterlinker 265. This first instance of a pair of identical primary reporters220 is also joined to a secondary reporter 280 by a first primaryreporter/secondary reporter linker 275A. A second instance of a pair ofidentical primary reporters 230 is also joined to the secondary reporter280 by a second primary reporter/secondary reporter linker 275B.

FIG. 2J schematically depicts an embodiment of a reporting construct(which can be described as C-A-B-D-C′) in which a first instance of apair of identical primary reporters 220 is joined to a membrane anchor270 by an interposing anchor/primary reporter linker 245. The membraneanchor 270 is also coupled to a cleavage site 260 via an anchor/cleavagesite linker 250. The cleavage site 260 is subsequently linked to asecondary reporter 280 by a cleavage site/secondary reporter linker 255.A second instance of a pair of identical primary reporters 230 is alsolinked to the secondary reporter, via a primary reporter/secondaryreporter linker 275.

FIG. 2K schematically depicts an embodiment of a reporting construct(which can be described as C-A-B-C′-D) in which a first instance of apair of identical primary reporters 220 is joined to a membrane anchor270 by an interposing anchor/primary reporter linker 245. The membraneanchor 270 is also coupled to a cleavage site 260 via an anchor/cleavagesite linker 250. The cleavage site 260 is subsequently linked to asecond instance of a pair of identical primary reporters 230 by ananchor/primary reporter linker 265. A secondary reporter 280 is alsolinked to the second instance of a pair of identical primary reporters230 by a primary reporter/secondary reporter linker 275.

In some embodiments a primary reporter is connected to a secondaryreporter by an intervening linker. In some of such embodiments, theprimary/secondary linker is selected to provide no significant (i.e.less than 5%) FRET between a primary reporter and a secondary reporter.For example, a primary/secondary reporter linker can be selected to havea length, geometry, and/or rigidity to maintain a distance and/ororientation between a primary reporter and a secondary reporter toreduce FRET to a negligible (i.e. <5%) amount. In other embodiments, aprimary/secondary reporter linker can be configured to provide a usefuldegree of FRET (i.e. >5%) between the primary reporter and a secondaryreporter.

FIG. 3 depicts a schematic of an exemplary assay 300 of the inventiveconcept. A cell with a cell membrane 310 and cytosol 315 has expressed aconstruct that includes a cell membrane anchoring portion 350, acleavage site 340, and two identical reporters 330, 335. Anchored to thecell membrane 310, the reporters 330, 335 produce a strong signal 360,365. To perform the assay the cell is exposed to an enzyme activity 320,which can act on the cleavage site 340. Hydrolysis of the peptidebackbone of the cleavage site releases the reporters into the cytosol315. Subsequent multiple degradative events result in degraded reporters330A, 335A that produce a modified signal 370, 375. In some embodimentsof the inventive concept the reporters are fluorescent proteins, and thefluorescence signal from the degraded fluorescent proteins is reduced.

FIG. 4 depicts an alternative assay 400 of the inventive concept. A cellwith a cell membrane 410, cytosol 415, and a vesicle 420 has expressed aconstruct that includes a vesicle membrane anchoring portion 450, acleavage site 440, and two reporters 430, 435. Anchored to the vesicle420, the reporters 430, 435 produce a strong signal 460, 465. To performthe assay the cell is exposed to an enzyme activity 425, which can acton the cleavage site 440. Hydrolysis of the cleavage site releases thereporters, resulting in release of the reporters 430, 435. Subsequentmultiple degradative event results in degraded reporters 430A, 435A thatproduce a modified signal 470, 475. In some embodiments of the inventiveconcept the reporters are fluorescent proteins, and the fluorescencesignal from the degraded fluorescent proteins is reduced.

In addition to utilizing different anchoring sites, assays of theinventive concept can use a variety of different testing protocols. Oneembodiment of such a testing protocol is shown in FIG. 5. Initially 510,a cell is provided that expresses a construct of the inventive concept.A baseline or first signal is acquired 520, then the cell is exposed tothe enzyme activity 530. For example, a sample containing an enzymeactivity (such as a BoNT) can be added to media containing the cells.After an incubation period a second signal can be detected 540 andsubsequently compared to the first signal 550. Such first and secondsignals can be instant measurements, mean measurements obtained overtime, and/or rate measurements.

An alternative embodiment of a test method of the inventive concept isshown in FIG. 6. Initially 610, a cell is provided that expresses aconstruct of the inventive concept. The cell is then exposed to theenzyme activity 620. For example, a sample containing an enzyme activity(such as a BoNT) can be added to media containing the cells. After afirst incubation period a baseline or first signal is detected 630 and,following a second incubation period a second signal is detected 640 andsubsequently compared to the first signal 650. Such first and secondsignals can be instant measurements, mean measurements obtained overtime, and/or rate measurements.

In another embodiment of the inventive concept, cells expressingconstructs as described above are exposed to one or more secondary dyes(for example a cell-binding dye such as a membrane dye or a nuclearstain/dye), that are separate from the construct and which can generatesignals that are independent of BoNT activity. Such secondary dyes canassociate with a membrane and/or nucleus of a cell in a fashion that isindependent of the presence of an analyte (for example, a BoNT or otherenzyme activity), and can be used to produce a baseline or referencesignal, which can be used for normalization. For example, a cellexpressing a construct as described above can be exposed to a dye thatassociates with the nucleus or plasma membrane of the cell, and in turnprovides a baseline fluorescent signal. In a preferred embodiment of theinventive concept such a secondary dye is selected such that theemission wavelengths of the membrane dye are distinguishable from thoseof a reporter fluorophore of the construct expressed in the cells. Insome embodiments of the inventive concept the secondary dye can beselected so that the range of effective excitation wavelengths overlapswith those of a reporter fluorophore of the construct, permittingsimultaneous excitation of both the secondary dye and the reporterfluorophore and hence simultaneous acquisition of a baseline signal anda reporter fluorophore signal. In other embodiments of the inventiveconcept a secondary dye can be selected so that the range of effectiveexcitation wavelengths does not overlap significantly with those of thereporter fluorophore, permitting selective excitation of baselinefluorescence. Examples of suitable secondary dyes include4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), the dye currentlyknown as CELLMASK™ deep red plasma membrane stain, and thenucleus-staining dye currently known as HOECHST3342™. In a preferredembodiment of the inventive concept the secondary dye is selected toprovide excitation and emission spectra that have little to no overlapwith the excitation and emission spectra of the reporter fluorophore ofthe construct, such that essentially no (i.e. less than about 5%) energytransfer occurs due to FRET. Examples of excitation and emission spectraof a suitable secondary dye (the dye currently known as CELLMASK™ deepred plasma membrane stain , indicated by “Cell Mask”) and a YFP reporterfluorophore are shown in FIG. 7. The inventors contemplate that othersuitable secondary dyes can include proteins (for example antibodies) orother macromolecules that have an affinity for the cell and have beenconjugated or complexed with fluorescent or other readily detectablemolecules.

Since association of secondary dyes with the cells is independent of thepresence of the analyte or activity of interest, they can provide abaseline signal that is an independent measure of cell number, density,and/or distribution. Such a baseline signal has considerable utility innormalization of the reporter signal obtained from cells in the courseof the performance of an assay of the inventive concept. For example,expressing a result of such an assay as a ratio between the measuredreporter signal from a reporter construct that is responsive to theanalyte or activity of interest and the measured baseline signal in theform of fluorescence from a membrane dye provides correction forvariation in the intensity of the reporter signal from test site to testsite due to differences in cell number, density, and/or distribution.This advantageously improves the precision of such assays, which in turnleads to an improvement in effective sensitivity. It should also beappreciated that such a baseline signal can be utilized to provide suchnormalization for reporter signals other than fluorescence.

EXAMPLE

1. As shown schematically in FIG. 8, cells transformed with anexpression vector encoding for a construct as described in FIG. 1A areseeded 810 into 96-well plates and incubated overnight at 37° C. with 5%CO₂.

2. Cells are then washed with cell culture media and then immediatelysubjected to BoNT 820 diluted into the cell culture media at 100 μl perwell (typically).

3. Cells are then incubated for 48 hours (typically) at 37° C. with 5%CO₂.

4. At the end of the 48 hour incubation period, the cells are stained830 with a secondary dye. Different secondary dyes may be applied usingdifferent protocols.

Protocol A: useful for DAPI and the dye currently known as HOECHST3342™.

1. 25 μl of 5 μM DAPI or 25 μg/ml the dye currently known asHOECHST3342™ in cell culture media is added directly to each well of the96-well plate to give a final concentration of 1 μM DAPI or 5 μg/ml thedye currently known as HOECHST3342™. Useful final concentrations of thenucleus dye range from 0.001-10 μor 0.1-50 μg/ml. Working solutionconcentrations may be adjusted appropriately.

2. Cells are incubated at 37° C., 5% CO₂ for 30 minutes (typically).

Protocol B: useful for the dye currently known as CELLMASK™ deep redplasma membrane stain.

1. A working solution of 0.625-10 μg/ml in Dulbecco's phosphate bufferedsaline is prepared (5 μg/ml is typical).

2. The BoNT containing cell culture media is removed from each well.

3. 50 μl of the membrane dye working solution is added directly to eachwell.

4. Cells are incubated at 37° C., 5% CO₂ for 20 minutes (typically).

After exposure to the membrane dye, the test plates are washed withphosphate buffered saline using an automated plate washer. The platesare then read on a fluorescence microplate reader using filters with theapproximate excitation and emission wavelengths for the fluorophore ofthe construct and the secondary dye used 840. For example, DAPI and thedye currently known as HOECHST3342™ can be characterized using anexcitation wavelength of about 345 nm and an emission wavelength ofabout 485 nm, while the dye currently known as CELLMASK™ deep red plasmamembrane stain can be characterized using an excitation wavelength ofabout 650 nm and an emission wavelength of about 680 nm. Measurements ofthe fluorophore of the construct are made independently of those of themembrane dye, using excitation and emission wavelengths characteristicof the fluorophore. For data analysis, directly excited constructfluorophore emission (i.e. the BoNT sensitive component) is normalized850, for example by dividing the emission of the construct fluorophoreby the secondary dye emission (i.e. the BoNT insensitive component).

Typical results of assays performed with reporting constructs asdescribed above in combination with secondary dyes are shown in FIGS.9A, 9B, and 9C. FIG. 9A shows typical uncorrected results fromcontacting cells containing a reporting construct with a protease (inthis example, the protease is BoNT/A and the construct includes acleavage site for BoNT/A. The limit of detection (LOD) in this instanceis 10 pM BoNT/A, and the limit of quantitation (LOQ) is 100 pM. FIG. 9Bshows the impact of normalization of the fluorescence data from FIG. 9Ausing emission data from a secondary dye applied to the same cells asdescribed in the above procedure. The LOD for the data shown in FIG. 9Bis reduced to 3 pM and the LOQ is reduced to 10 pM. FIG. 9C showsphotomicrographs of two wells containing cells having BoNT/A responsiveconstructs by (from left to right) brightfield, fluorescence microscopyat the emission wavelength of a fluorophore of the construct, andfluorescence microscopy at the emission wavelength of the secondary dyeapplied to the cells. The well in the upper series of photomicrographswas not exposed to BoNT/A; the well in the lower series ofphotomicrographs was exposed to 1 nM BoNT/A. The loss of fluorescencefrom the fluorophore of the construct is evident in the 1 nM BoNT/A well(i.e. the lower series). In contrast, emission from the secondary dye ismaintained at a similar, albeit distinct and different, level anddistribution from that of the well that was not exposed to BoNT/A (i.e.the upper series)

Variations of these protocols can also be effective. For example, thesecondary dyes can be applied prior to contacting the cells with theBoNT, essentially simultaneously with contacting the cells with theBoNT, or at a time interval of less than 48 hours following contactingthe cells with the BoNT. Similarly, additional wash steps prior to andfollowing exposure of the cells to the secondary dyes are contemplated.

It should be appreciated that assays of the inventive concept rely onstraightforward fluorescence measurements of fluid volumes rather thanimaging and/or analysis of individual cells. As such they can beperformed using a simple fluorometer (for example, a microplatefluorometer) and are advantageously highly amenable to adaptation toautomation and high throughput screening processes. Data analysis issimilarly straightforward, as it does not involve processor-intensiveimage processing tasks such as cell enumeration, identification ofindividual cells, and the identification of fluorescence localized inspecific subcellular regions or compartments.

In addition to providing a baseline signal for data normalizationpurposes, such secondary dyes can serve other purposes. For example abaseline signal value can be established below which cell numbers areconsidered insufficient to provide an accurate assay result, permittingdata from such a test site to be flagged or discarded. Similarly, abaseline signal value can be established above which cell numbers areconsidered too high to provide an accurate assay result (for example,due to optical limitations in systems utilized to characterizefluorescence). Inclusion of such secondary dyes with specific reagentsthat are added during the course of an assay can also be used to verifythat such reagents were actually delivered to a test site during theassay process, for example to verify that automated assay systems areperforming properly.

In preferred embodiments, the enzyme activity being characterized isassociated with botulinum toxin, and the cleavage sequence isappropriately matched. Within the context of this application, a BoNTcan be defined as a native or modified BoNT that is capable of cleavinga SNARE protein sequence or a portion of a SNARE protein sequence. Forexample, the BoNT/A, E, and C cleave SNAP-25 and BoNT/B, D, F, G cleavessynaptobrevin (Syb), at single but different sites. BoNT/C also cleavessyntaxin in addition to SNAP-25. Consequently, constructs for thecharacterization of BoNT/A, E, and C can include cleavage sitessequences that include all or a portion of SNAP-25. Similarly,constructs for the characterization of BoNT B, D, F, and G can includecleavage sites sequences that include all or portions of the respectivesusceptible regions of synaptobrevin. Alternatively, BoNT/C activitycould be characterized utilizing constructs that include cleavage siteswith sequences derived from all or part of syntaxin.

Contemplated cleavage site sequences can advantageously comprise a SNAREprotein, motif, or mutein. “Muteins” of a protein should be interpretedherein as having at least 30% identity with a corresponding nativeprotein, including for example compositions having at least 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identitywith the native protein. Variations from identity can comprise any ormore of additions, deletions and substitutions. Contemplated muteinsinclude fragments, truncates and fusion proteins.

It is further contemplated that cells of the inventive concept can bemodified to express two or more constructs. Such constructs could, forexample, be distinguished by the emission spectra of their respectivereporters and provide essentially independent and simultaneousmeasurements of different enzyme activities. Alternatively, suchconstructs could measure the activities of the same enzyme withdifferent substrate sequences. For example, a first construct couldinclude a cleavage site derived from SNAP-25 and a second constructcould include a cleavage site derived from syntaxin, with both beingused for characterizing BoNT/C activity. In such an embodimentcomparison of the results from both constructs can improve accuracy,dynamic range, and/or specificity.

Another embodiment of the inventive concept is a kit that incorporates asecondary reporter. Such a kit can contain cells that express anappropriate detecting construct, as described above, and a secondary dye(for example, a membrane dye). Optionally, such a kit can includedirections for a user to perform the assay. In some embodiments such akit can include control or calibration materials that include a suitablecell culture media and an enzyme activity corresponding to the enzymeactivity of the sample to be characterized. In this context, a controlsample is understood to be a sample used to verify assay performance,and a calibration sample is understood to be a sample used to calibratethe output of an assay to provide a quantitative or qualitative result.For example, of a sample suspected of containing a BoNT is to becharacterized, such control and/or calibration samples could include acorresponding BoNT. In some embodiments such control and/or calibrationsamples can be provided pre-mixed and essentially ready for use. Inother embodiments (for example, due to stability factors) such controland/or calibrator samples can be provided as a first container of asuitable culture media and a second container of a stock solution of theenzyme activity. In such embodiments the first and second containers mayrequire different shipping and/or storage conditions, and as such may beshipped and/or stored separately while remaining part of the same kit.

Other embodiments of the inventive concept include cell-free assaysutilizing the constructs described above. Such an assay could, forexample, utilize a cell-free vesicle suspension in which vesicles thatcarry one or more sites suitable for interacting with a membraneanchoring portion of a construct. Such vesicles, along with a constructof the inventive concept can be suspended in a medium that includes aprotease or similar enzyme capable of hydrolyzing a reporter, such thatcleavage of a linker portion of the construct would release reportersinto the media for hydrolysis. Alternatively, sites recognized by ananchoring portion of a construct can be linked to an appropriately sizedmicroparticle with a suitable surface chemistry. Such microparticles cancarry steric blockers, for example high molecular weight dextrans orpolyacrylates, that permit Botulinum toxins to access the microparticlesurface while hindering the access of proteases or similar enzymes.Towards that end, proteases or similar enzymes can be provided in highmolecular weight forms (for example, as polymers or as conjugates ofhigh molecular weight molecules) in order to enhance such selectivity.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A reporting construct for characterizing anenzyme activity comprising: a membrane anchoring domain comprising afirst peptide that forms a complex with a membrane of a cell; a reporterdomain comprising first occurrence of a second peptide that produces afirst signal at a first wavelength and a second occurrence of the secondpeptide that produces a second signal at the first wavelength, whereinthe reporter domain produces an aggregate signal that is a summation ofthe first signal and the second signal; a cleavage site comprising athird peptide, the cleavage site interposed between the membraneanchoring domain and at least two of the two or more occurrences of thereporter domain, wherein the third peptide is selected to undergo acleavage event upon exposure to the enzyme activity, wherein theaggregate signal is observable in the absence of either one of the firstsignal or the second signal.
 2. The reporting construct of claim 1,wherein the first occurrence of the second peptide is susceptible to afirst degradative event in the cell cytosol and the second occurrence ofthe second peptide is susceptible to a second degradative event in thecell cytosol following a cleavage event, wherein the first degradativeevent results in the loss of the production of the first signal and thesecond degradative event results in the loss of the production of thesecond signal.
 3. The reporting construct of claim 1, wherein the secondpeptide is a fluorescent protein.
 4. The reporting construct of claim 1,further comprising a linker peptide interposed between the firstoccurrence of the second peptide and the second occurrence of the secondpeptide.
 5. The reporting construct of claim 1, wherein the secondpeptide has at least 80% sequence identity to SEQ ID NO.
 10. 6. Thereporting construct of claim 1, wherein the third peptide is a SNAREprotein or a fragment thereof.
 7. The reporting construct of claim 1further comprising an auxiliary reporting domain that provides a signalat a second wavelength, wherein the second wavelength is distinguishablefrom the first wavelength.
 8. The reporting construct of claim 1,wherein the reporting construct shows reduced selectivity bias betweenthe first enzyme activity and a second enzyme activity relative to ananalogous reporting construct that includes a single occurrence of thesecond peptide, wherein the third peptide is selected to undergo acleavage event upon exposure to either of the first enzyme activity orthe second enzyme activity.
 9. The reporting construct of claim 9,wherein the first enzyme activity is associated with BoNT/A and thesecond enzyme activity is associated with BoNT/E.
 10. The reportingconstruct of claim 1, wherein the reporting construct does not exhibituseful levels of FRET.
 11. A method of characterizing a first enzymeactivity comprising: providing a cell that expresses a reportingconstruct comprising a membrane anchoring domain comprising a firstpeptide that forms a complex with a membrane of a cell, a reporterdomain comprising a first occurrence of a second peptide that produces afirst signal at a first wavelength and a second occurrence of the secondpeptide that produces a second signal at the first wavelength whereinthe reporter domain produces an aggregate signal that is a summation ofthe first signal and the second signal, and a cleavage site comprising athird peptide, the cleavage site interposed between the membraneanchoring domain and at least two of the two or more occurrences of thereporter domain and wherein the third peptide is selected to undergo acleavage event upon exposure to the first enzyme activity; contactingthe cell with a sample suspected of including the first enzyme activity;and observing a decrease in the detectable signal when the sampleincludes the first enzyme activity, wherein the aggregate signal isobservable in the absence of either one of the first signal or thesecond signal.
 12. The method of claim 11, wherein the second peptide isa fluorescent protein.
 13. The method of claim 11, wherein the methodshows reduced selectivity between the first enzyme activity and a secondenzyme activity relative to an assay utilizing an analogous reportingconstruct that includes a single occurrence of the second peptide,wherein the third peptide is selected to undergo a cleavage event uponexposure to either of the first enzyme activity or the second enzymeactivity.
 14. The method of claim 13, wherein the first enzyme activityis associated with BoNT/A and the second enzyme activity is associatedwith BoNT/E.
 15. The method of claim 11, wherein the second peptide hasat least 80% sequence identity to SEQ ID NO.
 10. 16. The method of claim11, wherein the third peptide is a SNARE protein or a fragment thereof.17. The method of claim 11 wherein the reporting construct furthercomprises a reference reporter that produces a reference signal, whereinthe reference signal is distinguishable from the detectable signal andis independent of the enzyme activity of the sample.
 18. The method ofclaim 17, wherein the reference signal is used to normalize theaggregate signal.
 19. The method of claim 11, further comprising thestep of contacting the cell with a cell dye, wherein the cell dye isselected to provide a dye signal that is distinguishable from theaggregate signal.
 20. The method of claim 19, wherein the dye signal isused to normalize the aggregate signal.
 21. The method of claim 19,wherein the cell is contacted with the cell dye prior to contacting thecell with the sample.
 22. The method of claim 19, wherein the cell iscontacted with the cell dye after the cell is contacted with the sample.23. The method of claim 19, wherein the cell is contacted with the celldye and the sample during the same time interval.
 24. The method ofclaim 11, wherein the reporting construct does not exhibit useful levelsof FRET.